Tissue analysis and kits therefor

ABSTRACT

This invention relates to methods of analyzing a tissue sample from a subject. In particular, the invention combines morphological staining and/or immunohistochemistry (IHC) with fluorescence in situ hybridization (FISH) within the same section of a tissue sample. The analysis can be automated or manual. The invention also relates to kits for use in the above methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/978,033, filed Oct. 29, 2004, now U.S. Pat. No. 7,129,051, which is acontinuation of U.S. application Ser. No. 10/351,067, filed Jan. 23,2003, now U.S. Pat. No. 6,905,830, which is a continuation of U.S.application Ser. No. 09/167,691, filed on Oct. 7, 1998, now U.S. Pat.No. 6,573,043, which applications are hereby incorporated herein byreference and from which applications priority is claimed under 35U.S.C. §120.

FIELD OF THE INVENTION

The present invention relates generally to the field of tissue analysis.Specifically, the invention combines morphological staining and/orimmunohistochemistry (IHC) with fluorescence in situ hybridization(FISH) within the same section of a tissue sample thereby allowing foraccurate and simplified prognostic, diagnostic, or research applicationson a subject's tissue sample. In addition, the invention provides kitsfor analysis of a tissue sample utilizing the present methods.

BACKGROUND OF THE INVENTION

Advancements in the understanding of genetics and developments intechnology and epidemiology have allowed for the correlation of geneticabnormalities with certain malignancies as well as risk assessment of anindividual for developing certain malignancies. However, most of themethodologies available for evaluation of tissue for the presence ofgenes associated with or predisposing an individual to a malignancy havewell-known drawbacks. For example, methods that require disaggregationof the tissue, such as Southern, Northern, or Western blot analysis, arerendered less accurate by dilution of the malignant cells by the normalor otherwise non-malignant cells that are present in the same tissue.Furthermore, the resulting loss of tissue architecture precludes theability to correlate malignant cells with the presence of geneticabnormalities in a context that allows morphological specificity. Thisissue is particularly problematic in tissue types known to beheterogeneous, such as in human breast carcinoma, where a significantpercentage of the cells present in any area may be non-malignant.

Fluorescence in situ hybridization (FISH) is a recently developed methodfor directly assessing the presence of genes in intact cells. FISH is anattractive means of evaluating paraffin-embedded tissue for the presenceof malignancy because it provides for cell specificity, yet overcomesthe cross-linking problems and other protein-altering effects caused byformalin fixation. FISH has historically been combined with classicalstaining methodologies in an attempt to correlate genetic abnormalitieswith cellular morphology [see e.g., Anastasi et al., Blood 77:2456-2462(1991); Anastasi et al., Blood 79:1796-1801 (1992); Anastasi et al.,Blood 81:1580-1585 (1993); van Lom et al., Blood 82:884-888 (1992);Wolman et al., Diagnostic Molecular Pathology 1(3): 192-199 (1992);Zitzelberger, Journal of Pathology 172:325-335 (1994)]. However, severalof these studies address hematological disorders where genetic changesare assessed in freshly fixed smears from bone marrow aspirates orperipheral blood specimens. Of those two studies where paraffin-embeddedtissue was analyzed, one involved evaluation of FISH and morphologicalstaining on separate, serial sections. In the other study, bothprocedures were performed on the same section, but morphologicalstaining was subsequent to evaluation by FISH. Use of serial sections inthis type of analysis increases the probability of error, especially inheterogeneous tissue such as breast tissue.

Immunohistochemical staining of tissue sections has been shown to be areliable method of assessing alteration of proteins in a heterogeneoustissue. Immunohistochemistry (IHC) techniques utilize an antibody toprobe and visualize cellular antigens in situ, generally by chromagenicor fluorescent methods. This technique excels because it avoids theunwanted effects of disaggregation and allows for evaluation ofindividual cells in the context of morphology. In addition, the targetprotein is not altered by the freezing process.

The HER2/neu gene encodes a protein product, often identified asp185^(HER2) The native p185^(HER2) protein is a membrane receptor-likemolecule with homology to the epidermal growth factor receptor (EGFR).Amplification and overexpression of HER2 in human breast cancer has beencorrelated with shorter disease-free interval and shorter overallsurvival in some studies [van de Vijer et al. New Eng. J. Med.317:1239(1988); Walker et al. Br. J. Cancer 60:426(1989); Tandon et al.J. Clin. Invest. 7:1120 (1989); Wright et al. Cancer Res. 49:2087(1989); McCann et al. Cancer Res 51:3296 (1991); Paterson et al. CancerRes. 51:556 (1991); and Winstanley et al. Br. J. Cancer 63:447 (1991)]but not in others [Zhou et al. Oncogene 4:105 (1989); Heintz et al. ArchPath Lab Med 114:160 (1990); Kury et al. Eur. J. Cancer 26:946 (1990);Clark et al. Cancer Res. 51:944 (1991); and Ravdin et al. J. Clin.Oncol. 12:467-74 (1994)].

In an initial evaluation of 103 patients with breast cancer, thosehaving more than three tumor cell positive axillary lymph nodes (nodepositive) were more likely to overexpress HER2 protein than patientswith less than three positive nodes [Slamon et al. Science 235:177(1987)]. In a subsequent evaluation of 86 node-positive patients withbreast cancer, there was a significant correlation among the extent ofgene amplification, early relapse, and short survival. HER2overexpression was determined using Southern and Northern blotting whichcorrelate with the HER2 oncoprotein expression evaluated by Westernblotting and IHC [Slamon et al. Science 235:177 (1987); Slamon et al.Science 244:707 (1989)]. The median period of survival was found to beapproximately 5-fold shorter in patients with more than five copies ofthe HER2 gene than in patients without gene amplification. Thiscorrelation was present even after correcting for nodal status and otherprognostic factors in multivariate analyses. These studies were extendedin 187 node-positive patients and indicated that gene amplification,increased amounts of mRNA (determined by Northern blotting), andincreased protein expression (determined immunohistochemically) werealso correlated with shortened survival time [Slamon et al. Science244:707 (1989)]. See also U.S. Pat. No. 4,968,603 to Slamon et al

Nelson et al. have compared HER2/neu gene amplification using FISH withimmunohistochemically determined overexpression in breast cancer [Nelsonet al. Modern Pathology 9 (1) 21A (1996)].

SUMMARY OF THE INVENTION

The present invention combines cellular morphological analysis withfluorescence in situ hybridization (FISH) to provide for a correlationof genetic abnormalities and cellular morphology within the same sectionof a subject's tissue sample. Accordingly, one may identify and score byFISH cancer cells (e.g. invasive ductal carcinoma cells) as distinctfrom other normal cells (e.g. stromal and inflamnatory elements found inthe biopsy). Alternatively, or additionally, the invention combinesimmunohistochemistry (IHC) with FISH to provide for a correlation ofgenetic abnormalities with protein expression in the same tissuesection.

Morphologic assessment, or evaluation of protein expression, in a tissueprior to quantitative FISH analysis provides for accurate, specificevaluation of that tissue in a timely and cost-efficient manner. Thus,there is a need in research, prognostic, and diagnostic applications fora method that can allow for morphologic and/or protein expressionanalyses followed by FISH assessment in a single tissue sample section,particularly when testing a heterogeneous tissue. The inventiondescribed in this disclosure offers these features.

Accordingly, in a first aspect the invention provides a method ofcorrelating cellular morphology with the presence of a cellular targetnucleic acid sequence in a section of a tissue sample comprising thefollowing steps:

(a) staining the section of tissue sample with a morphological stain;

(b) determining cellular morphology in the section of tissue sample;

(c) hybridizing a first fluorescently labeled nucleic acid probe to thetarget nucleic acid sequence in the same section of tissue sample;

(d) detecting the presence of the first nucleic acid probe in thesection of tissue sample; and

(e) correlating step (b) with step (d).

In an alternative embodiment, the invention pertains to a method ofcorrelating the presence of a cellular target protein with the presenceof a cellular target nucleic acid sequence in a section of a tissuesample comprising the following steps:

(a) contacting the section of sample tissue with an antibody whichspecifically binds to the target protein;

(b) determining binding of the antibody to the section of tissue sample;

(c) hybridizing a fluorescently labeled nucleic acid probe to the targetnucleic acid sequence in the same section of tissue sample;

(d) detecting the presence of the labeled nucleic acid probe in thesection of tissue sample; and

(e) correlating step (b) with step (d).

Additionally, the invention provides a kit comprising (a) amorphological stain; (b) a fluorescently labeled probe complementary toa genetic abnormality; and (c) instructions for applying the stain (a)and probe (b) to the same section of tissue sample.

Moreover, a kit is provided comprising: (a) a primary antibody whichspecifically binds a cellular target protein; (b) a fluorescentlylabeled probe complementary to a genetic abnormality; and (c)instructions for applying the antibody (a) and probe (b) to the samesection of tissue sample.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the effect of HEMA 3® staining on the ability to score FISHin formalin fixed, paraffin-embedded cells harvested from each of threecell lines of known HER2/neu amplification status; SKBR3 (highlyamplified HER2), MDA175 (barely amplified HER2), and MDA231(non-amplified HER2).

FIG. 2 shows the effect of HEMA 3® morphologic stain on FISH withrespect to tumor biopsy sections. Duplicate sections from 13 tumorspecimens were analyzed for HER2 and chromosome 17 (Chr 17) by FISHeither with or without prior staining for morphologic analysis prior toFISH. The same area of tumor was scored for FISH on each section.

FIG. 3 shows the mean HER2/neu:Chr 17 ratio for blinded vs non-blindedassessment of normal tissue.

FIG. 4 shows the mean HER2/neu:Chr 17 ratio for blinded vs non-blindedassessment of tumor tissue.

FIG. 5 shows the relationship between the scoring systems used forHER2/neu amplification by FISH and overexpression by IHC.

DETAILED DESCRIPTION OF THE INVENTION

Before the present methods, kits and uses therefor are described, it isto be understood that this invention is not limited to the particularmethodology, protocols, cell lines, animal species or genera,constructs, and reagents described as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “agenetic alteration” includes a plurality of such alterations andreference to “a probe” includes reference to one or more probes andequivalents thereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devicesand materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited.

Publications cited herein are cited for their disclosure prior to thefiling date of the present application. Nothing here is to be construedas an admission that the inventors are not entitled to antedate thepublications by virtue of an earlier priority date or prior date ofinvention. Further the actual publication dates may be different fromthose shown and require independent verification.

Definitions

By “subject” or “patient” is meant any single subject for which therapyis desired, including humans, cattle, dogs, guinea pigs, rabbits,chickens, insects and so on. Also intended to be included as a subjectare any subjects involved in clinical research trials not showing anyclinical sign of disease, or subjects involved in epidemiologicalstudies, or subjects used as controls.

By “tissue sample” is meant a collection of similar cells obtained froma tissue of a subject or patient, preferably containing nucleated cellswith chromosomal material. The four main human tissues are (1)epithelium; (2) the connective tissues, including blood vessels, boneand cartilage; (3) muscle tissue; and (4) nerve tissue. The source ofthe tissue sample may be solid tissue as from a fresh, frozen and/orpreserved organ or tissue sample or biopsy or aspirate; blood or anyblood constituents; bodily fluids such as cerebral spinal fluid,amniotic fluid, peritoneal fluid, or interstitial fluid; cells from anytime in gestation or development of the subject. The tissue sample mayalso be primary or cultured cells or cell lines. The tissue sample maycontain compounds which are not naturally intermixed with the tissue innature such as preservatives, anticoagulants, buffers, fixatives,nutrients, antibiotics, or the like. In one embodiment of the invention,the tissue sample is “non-hematologic tissue” (i.e. not blood or bonemarrow tissue).

For the purposes herein a “section” of a tissue sample is meant a singlepart or piece of a tissue sample, e.g. a thin slice of tissue or cellscut from a tissue sample. It is understood that multiple sections oftissue samples may be taken and subjected to analysis according to thepresent invention, provided that it is understood that the presentinvention comprises a method whereby the same section of tissue sampleis analyzed at both morphological and molecular levels, or is analyzedwith respect to both protein and nucleic acid.

By “correlate” or “correlating” is meant comparing, in any way, theperformance and/or results of a first analysis with the performanceand/or results of a second analysis. For example, one may use theresults of a first analysis in carrying out the second analysis and/orone may use the results of a first analysis to determine whether asecond analysis should be performed and/or one may compare the resultsof a first analysis with the results of a second analysis. With respectto the embodiment of morphological analysis followed by FISH, one mayuse the results obtained upon morphological staining to determinearea(s) of a tissue section which are normal and/or area(s) which arecancerous. Thus, histologically normal area(s) in a heterogeneous tumorbiopsy may be used as internal normal control(s). In relation to IHCcombined with FISH, one may use the results of IHC to determine whetherFISH should be performed and/or one may compare the level of proteinexpression with gene amplification to further characterize a tumorbiopsy (e.g. to compare HER2 protein expression with HER2 geneamplification).

By “nucleic acid” is meant to include any DNA or RNA. For example,chromosomal, mitochondrial, viral and/or bacterial nucleic acid presentin tissue sample. The term “nucleic acid” encompasses either or bothstrands of a double stranded nucleic acid molecule and includes anyfragment or portion of an intact nucleic acid molecule.

By “gene” is meant any nucleic acid sequence or portion thereof with afunctional role in encoding or transcribing a protein or regulatingother gene expression. The gene may consist of all the nucleic acidsresponsible for encoding a functional protein or only a portion of thenucleic acids responsible for encoding or expressing a protein. Thenucleic acid sequence may contain a genetic abnormality within exons,introns, initiation or termination regions, promoter sequences, otherregulatory sequences or unique adjacent regions to the gene.

By “genetic abnormality” is meant a deletion, substitution, addition,translocation, amplification and the like relative to the normal nativenucleic acid content of a cell of a subject.

By “disease gene” is meant a gene that results in altered proteinproduct (i.e., protein different from native protein in terms ofsequence, structure and/or amount expressed) and results in a disease.

By “deletion” is meant the absence of all or part of a gene.

By “amplification” is meant the presence of one or more extra genecopies in a chromosome complement.

The word “label” when used herein refers to a compound or compositionwhich is conjugated or fused directly or indirectly to a reagent such asa nucleic acid probe or an antibody and facilitates detection of thereagent to which it is conjugated or fused. The label may itself bedetectable (e.g., radioisotope labels or fluorescent labels) or, in thecase of an enzymatic label, may catalyze chemical alteration of asubstrate compound or composition which is detectable.

The term “fluorescently labeled nucleic acid probe” refers to a probecomprising (1) a nucleic acid sequence capable, of hybridizing with atarget nucleic acid sequence and (2) a fluorescent label.

By “morphological stain” is meant a dye that stains different cellularcomponents, in order to facilitate identification of cell type and/ordisease status by light microscopy. Preferably, the morphological stainis readily distinguishable from any label used in the FISH analysis,e.g., a stain which will not autofluoresce at the same wavelength as thefluorochrome used in the FISH analysis.

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies, polyclonal antibodies, multispecificantibodies (e.g., bispecific antibodies), and antibody fragments so longas they bind specifically to a target antigen.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations that typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al., Nature256:495 (1975), or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol.Biol. 222:581-597 (1991), for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity [U.S. Pat. No. 4,816,567;and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)].

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, Fv framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

The term “primary antibody” herein refers to an antibody which bindsspecifically to the target protein antigen in a tissue sample. A primaryantibody is generally the first antibody used in an immunohistochemicalprocedure. In one embodiment, the primary antibody is the only antibodyused in an IHC procedure.

The term “secondary antibody” herein refers to an antibody which bindsspecifically toga primary antibody, thereby forming a bridge between theprimary antibody and a subsequent reagent, if any. The secondaryantibody is generally the second antibody used in an immunohistochemicalprocedure.

Unless indicated otherwise, the terms “HER2”, “p185^(HER2)” and “ErbB2”when used herein refer to human HER2 protein or a portion thereof and“HER2”, “HER2/neu” and “c-erbB2” refer to the human HER2 gene or aportion thereof. The human HER2 gene and HER2 protein are, for example,described in Semba et al., PNAS (USA) 82:6497-6501 (1985) and Yamamotoet al. Nature 319:230-234 (1986) (Genebank accession number X03363).

Dual Analysis Method

The present invention relates to a method which combines morphologicalstaining and/or immunohistochemical analysis with fluorescence in situhybridization (FISH) within the same section of a tissue sample. Thepresent methods allow for a highly accurate and simplified method ofcorrelating morphological criteria or protein expression with geneticabnormalities. Because many tissue types, such as breast tissue, arecharacterized by significant cellular heterogeneity, inaccurate resultsmay be obtained when serial sections from a tissue block are analyzed bytwo different methods. According to the present invention, both types ofanalysis are carried out on the same tissue section, thereby reducingerror when analyzing such heterogeneous tissue. As evidenced by thefollowing, the method of this application is useful in a variety ofprognostic, diagnostic and research applications. Also disclosed arekits for use in the disclosed methods.

Sample Preparation

Any tissue sample from a subject may be used. Examples of tissue samplesthat may be used include, but are not limited to, breast, prostate,ovary, colon, lung, endometrium, stomach, salivary gland or pancreas.The tissue sample can be obtained by a variety of procedures including,but not limited to surgical excision, aspiration or biopsy. The tissuemay be fresh or frozen. In one embodiment, the tissue sample is fixedand embedded in paraffin or the like.

The tissue sample may be fixed (i.e. preserved) by conventionalmethodology [See e.g., “Manual of Histological Staining Method of theArmed Forces Institute of Pathology,” 3^(rd) edition (1960) Lee G. Luna,HT (ASCP) Editor, The Blakston Division McGraw-Hill Book Company, NewYork; The Armed Forces Institute of Pathology Advanced LaboratoryMethods in Histology and Pathology (1994) Ulreka V. Mikel, Editor, ArmedForces Institute of Pathology, American Registry of Pathology,Washington, D.C.]. One of skill in the art will appreciate that thechoice of a fixative is determined by the purpose for which the tissueis to be histologically stained or otherwise analyzed. One of skill inthe art will also appreciate that the length of fixation depends uponthe size of the tissue sample and the fixative used. By way of example,neutral buffered formalin, Bouin's or paraformaldehyde, may be used tofix a tissue sample.

Generally, the tissue sample is first fixed and is then dehydratedthrough an ascending series of alcohols, infiltrated and embedded withparaffin or other sectioning media so that the tissue sample may besectioned. Alternatively, one may section the tissue and fix thesections obtained. By way of example, the tissue sample may be embeddedand processed in paraffin by conventional methodology (See e.g., “Manualof Histological Staining Method of the Armed Forces Institute ofPathology”, supra). Examples of paraffin that may be used include, butare not limited to, Paraplast, Broloid, and Tissuemay. Once the tissuesample is embedded, the sample may be sectioned by a microtome or thelike (See e.g., “Manual of Histological Staining Method of the ArmedForces Institute of Pathology”, supra). By way of example for thisprocedure, sections may range from about three microns to about fivemicrons in thickness. Once sectioned, the sections may be attached toslides by several standard methods. Examples of slide adhesives include,but are not limited to, silane, gelatin, poly-L-lysine and the like. Byway of example, the paraffin embedded sections may be attached topositively charged slides and/or slides coated with poly-L-lysine.

If paraffin has been used as the embedding material, the tissue sectionsare generally deparaffinized and rehydrated to water. The tissuesections may be deparaffinized by several conventional standardmethodologies. For example, xylenes and a gradually descending series ofalcohols may be used (See e.g., “Manual of Histological Staining Methodof the Armed Forces Institute of Pathology”, supra). Alternatively,commercially available deparaffinizing non-organic agents such asHemo-De® (CMS, Houston, Tex.) may be used.

Morphological Staining

After deparaffinization, the sections mounted on slides may be stainedwith a morphological stain for evaluation. The morphological stain to beused in the instant method provides for accurate morphologicalevaluation of a tissue section and also allows for accuratequantification of fluorescently labeled (e.g., SPECTRUM ORANGE® andSPECTRUM GREEN®) nucleic acid probes when the sections are subsequentlyprocessed for FISH. Many morphological stains fluoresce when illuminatedby light of the same wavelength required to visualize the probefluorophore of interest. The disclosed method solves this problem.Generally, the section is stained with one or more dyes each of whichdistinctly stains different cellular components. In a preferredembodiment xanthine dye or the functional equivalent thereof and/or athiazine dye or the functional equivalent thereof are used to enhanceand make distinguishable the nucleus, cytoplasm, and “granular”structures within each. Such dyes are commercially available and oftensold as sets. By way of example, HEMA 3® (CMS, Houston, Tex.) stain setcomprises xanthine dye and thiazine dye. Methylene blue may also beused. Examples of other morphological stains that may be used on theinstant method include, but are not limited to, dyes that do notsignificantly autofluoresce at the same wavelength as fluorescentlabel(s) used for the subsequent FISH analysis. For example, where thefluorescent labels used for the FISH are SPECTRUM ORANGE® and SPECTRUMGREEN®, the morphological stain preferably does not significantlyfluoresce at about 509/538 (peak excitation/emission) and/or about559/588 (peak excitation/emission). One of skill in the art willappreciate that staining may be optimized for a given tissue byincreasing or decreasing the length of time the slides remain in thedye.

After staining, the tissue section may be analyzed by standardtechniques of microscopy. Generally, a pathologist or the like assessesthe tissue for the presence of abnormal or normal cells or a specificcell type and provides the loci of the cell types of interest. Thus, forexample, in a study correlating. HER2/neu amplification in breastcancer, a pathologist or the like would review the slides and identifynormal breast cells and abnormal breast cells (See e.g. Example 2).

Any means of defining the loci of the cells of interest may be used(e.g., coordinates on an X-Y axis). Generally, after light microscopyand prior to the FISH procedure, the slides are destained byconventional methodology. The present method provides an advantage overthe prior procedures in the art in that no separate destaining procedureis required prior to FISH. Avoidance of a destaining step is actuallypreferred in order to protect the integrity of the DNA for in situhybridization.

Immunohistochemistry (IHC)

Prior to FISH, the tissue section may be subjected to IHC. IHC may beperformed in combination with morphological staining as discussed in thepreceding section (either prior to, but preferably thereafter).

Two general methods of IHC are available; direct and indirect assays.According to the first assay, binding of antibody to the target antigenis determined directly. This direct assay uses a labeled reagent, suchas a fluorescent tag or an enzyme-labeled primary antibody, which can bevisualized without further antibody interaction. In a typical indirectassay, unconjugated primary antibody binds to the antigen and then alabeled secondary antibody binds to the primary antibody. Where thesecondary antibody is conjugated to an enzymatic label, a chromagenic orfluorogenic substrate is added to provide visualization of the antigen.Signal amplification occurs because several secondary antibodies mayreact with different epitopes on the primary antibody.

The primary and/or secondary antibody used for immunohistochemistrytypically will be labeled with a detectable moiety. Numerous labels areavailable which can be generally grouped into the following categories:

(a) Radioisotopes, such as ³⁵S, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I. The antibodycan be labeled with the radioisotope using the techniques described inCurrent Protocols in Immunology, Volumes 1 and 2, Coligen et al., Ed.Wiley-Interscience, New York, N.Y., Pubs. (1991) for example andradioactivity can be measured using scintillation counting.

(b) Colloidal gold particles.

(c) Fluorescent labels including, but are not limited to, rare earthchelates (europium chelates), Texas Red, rhodamine, fluorescein, dansyl,Lissamine, umbelliferone, phycocrytherin, phycocyanin, or commerciallyavailable fluorophores such SPECTRUM ORANGE® and SPECTRUM GREEN® and/orderivatives of any one or more of the above. The fluorescent labels canbe conjugated to the antibody using the techniques disclosed in CurrentProtocols in Immunology, supra, for example. Fluorescence can bequantified using a fluorimeter.

(d) Various enzyme-substrate labels are available and U.S. Pat. No.4,275,149 provides a review of some of these. The enzyme generallycatalyzes a chemical alteration of the chromogenic substrate that can bemeasured using various techniques. For example, the enzyme may catalyzea color change in a substrate, which can be measuredspectrophotometrically. Alternatively, the enzyme may alter thefluorescence or chemiluminescence of the substrate. Techniques forquantifying a change in fluorescence are described above. Thechemiluminescent substrate becomes electronically excited by a chemicalreaction and may then emit light which can be measured (using achemiluminometer, for example) or donates energy to a fluorescentacceptor. Examples of enzymatic labels include luciferases (e.g. fireflyluciferase and bacterial luciferase; U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase,β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to antibodies are described in O'Sullivan et al.,Methods for the Preparation of Enzyme-Antibody Conjugates for use inEnzyme Immunoassay, in Methods in Enzym. (ed J. Langbne & H. VanVunakis), Academic press, New York, 73:147-166 (1981).

Examples of enzyme-substrate combinations include, for example:

(i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as asubstrate, wherein the hydrogen peroxidase oxidizes a dye precursor[e.g., orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethyl benzidinehydrochloride (TMB)];

(ii) alkaline phosphatase (AP) with para-Nitrophenyl phosphate aschromogenic substrate; and

(iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g.,p-nitrophenyl-β-D-galactosidase) or fluorogenic substrate (e.g.,4-methylumbelliferyl-β-D-galactosidase).

Numerous other enzyme-substrate combinations are available to thoseskilled in the art. For a general review of these, see U.S. Pat. Nos.4,275,149 and 4,318,980.

Sometimes, the label is indirectly conjugated with the antibody. Theskilled artisan will be aware of various techniques for achieving this.For example, the antibody can be conjugated with biotin and any of thefour broad categories of labels mentioned above can be conjugated withavidin, or vice versa. Biotin binds selectively to avidin and thus, thelabel can be conjugated with the antibody in this indirect manner.Alternatively, to achieve indirect conjugation of the label with theantibody, the antibody is conjugated with a small hapten and one of thedifferent types of labels mentioned above is conjugated with ananti-hapten antibody. Thus, indirect conjugation of the label with theantibody can be achieved.

Aside from the sample preparation procedures discussed above, furthertreatment of the tissue section prior to, during or following IHC may bedesired, For example, epitope retrieval methods, such as heating thetissue sample in citrate buffer may be carried out [see, e.g., Leong etal. Appl. Immnunohistochem. 4(3):201 (1996)].

Following an optional blocking step, the tissue section is exposed toprimary antibody for a sufficient period of time and under suitableconditions such that the primary antibody binds to the target proteinantigen in the tissue sample. Appropriate conditions for achieving thiscan be determined by routine experimentation.

The extent of binding of antibody to the sample is determined by usingany one of the detectable labels discussed above. Preferably, the labelis an enzymatic label (e.g. HRPO) which catalyzes a chemical alterationof the chromogenic substrate such as 3,3′-diaminobenzidine chromogen.Preferably the enzymatic label is conjugated to antibody which bindsspecifically to the primary antibody (e.g. the primary antibody israbbit polyclonal antibody and secondary antibody is goat anti-rabbitantibody).

Specimens thus prepared may be mounted and coverslipped. Slideevaluation is then determined, e.g. using a microscope.

Where the antigen is HER2 protein, staining intensity criteria may beevaluated as follows:

TABLE 1 HER2 Protein Staining Intensity Criteria Staining Pattern ScoreNo staining is observed in tumor cells. 0 A faint/barely perceptiblemembrane staining is detected in 1+ tumor cells. The cells are onlystained in part of their membrane. A weak to moderate complete membranestaining is 2+ observed in tumor cells. A moderate to strong completemembrane staining is 3+ observed in tumor cells.

In this embodiment of the invention, it may be desirable to select asubgrouping of the tissue samples subjected to IHC for further analysisby FISH. For example, tissue samples with 1+ and 2+ scores, andespecially, the 2+ subgroup may be subjected to further FISH asexplained below.

Fluorescence In Situ Hydridization (FISH)

In situ hybridization is generally carried out on cells or tissuesections fixed to slides. In situ hybridization may be performed byseveral conventional methodologies [See for e.g. Leitch et al. In SituHybridization: a practical guide, Oxford BIOS Scientific Publishers,Micropscopy handbooks v. 27 (1994)]. In one in situ procedure,fluorescent dyes [such as fluorescein isothiocyanate (FITC) whichfluoresces green when excited by an Argon ion laser] are used to label anucleic acid sequence probe which is complementary to a targetnucleotide sequence in the cell. Each cell containing the targetnucleotide sequence will bind the labeled probe producing a fluorescentsignal upon exposure, of the cells to a light source of a wavelengthappropriate for excitation of the specific fluorochrome used.

Various degrees of hybridization stringency can be employed. As thehybridization conditions become more stringent, a greater degree ofcomplementarity is required between the probe and target to form andmaintain a stable duplex. Stringency is increased by raisingtemperature, lowering salt concentration, or raising formamideconcentration. Adding dextran sulfate or raising its concentration mayalso increase the effective concentration of labeled probe to increasethe rate of hybridization and ultimate signal intensity. Afterhybridization, slides are washed in a solution generally containingreagents similar to those found in the hybridization solution withwashing time varying from minutes to hours depending on requiredstringency. Longer or more stringent washes typically lower nonspecificbackground but run the risk of decreasing overall sensitivity. Exemplaryin situ hybridization conditions for detecting HER2/neu amplification inbreast tissue are shown in Example 2.

Probes used in the FISH analysis may be either RNA or DNAoligonucleotides or polynucleotides and may contain not only naturallyoccurring nucleotides but their analogs like digoxygenin dCTP, biotindcTP 7-azaguanosine, azidothymidine, inosine, or uridine. Other usefulprobes include peptide probes and analogues thereof, branched gene DNA,peptidometics, peptide nucleic acid (PNA) and/or antibodies.

Probes should have sufficient complementarity to the target nucleic acidsequence of interest so that stable and specific binding occurs betweenthe target nucleic acid sequence and the probe. The degree of homologyrequired for stable hybridization varies with the stringency of thehybridization medium and/or wash medium. Preferably, completelyhomologous probes are employed in the present invention, but persons ofskill in the art will readily appreciate that probes exhibiting lesserbut sufficient homology can be used in the present invention [see fore.g. Sambrook, J., Fritsch, E. F., Maniatis, T., Molecular Cloning ALaboratory Manual, Cold Spring Harbor Press, (1989)].

One of skill in the art will appreciate that the choice of probe willdepend on the genetic abnormality of interest. Genetic abnormalitiesthat can be detected by this method include, but are not limited to,amplification, translocation, deletion, addition and the like. Examplesof amplification include, but are not limited to, HER2/neu in breast andovarian cancer, N-myc in neuroblastoma, C-myc in small cell lung cancer.Examples of abnormal chromosome number include, but are not limited to,trisomy 8 in leukemia, monosomy 7 in myloproliferative disorders, andtrisomy 12 in chronic lymphoblastic leukemia. Examples of translocationsinclude, but are not limited to, bcr/abl [t (9;22)] translocation inchronic mylogenous leukemia and the t (15;17) translocation FAB-M3(acute promyelocytic leukemia). Examples of deletions include EGFR vIIIand p53. By way of example for evaluating HER2/neu amplification a probespanning a 140 kb region on the long arm of chromosome 17 containing theHER2/neu gene (17q 11.2-17q12) may be used. A probe for the -satellitesequences in the centromeric region of chromosome 17(D1721) may be usedto evaluate for aneusomy of chromosome 17 as a source or cause forHER2/neu amplification. For example, a cocktailed version of theseprobes may be obtained from Vysis, Inc. where each probe is directlylabeled with easily distinguishable fluorophores, such as SPECTRUMORANGE® and SPECTRUM GREEN®.

Probes may also be generated and chosen by several means including, butnot limited to, mapping by in situ hybridization, somatic cell hybridpanels, or spot blots of sorted chromosomes; chromosomal linkageanalysis; or cloned and isolated from sorted chromosome libraries fromhuman cell lines or somatic cell hybrids with human chromosomes,radiation somatic cell hybrids, microdissection of a chromosome region,or from yeast artificial chromosomes (YACs) identified by PCR primersspecific for a unique chromosome locus or other suitable means like anadjacent YAC clone. Probes may be genomit DNA, cDNA, or RNA cloned in aplasmid, phage, cosmid, YAC, Bacterial Artificial Chromosomes (BACs),viral vector, or any other suitable vector. Probes may be cloned orsynthesized chemically by conventional methods. When cloned, theisolated probe nucleic acid fragments are typically inserted into avector, such as lambda phage, pBR322, M13, or vectors containing the SP6or T7 promoter and cloned as a library in a bacterial host. [See fore.g. Sambrook, J., Fritsch, E. F., Maniatis, T., Molecular Cloning ALaboratory Manual, Cold Spring Harbor Press, (1989)].

Probes are preferably labeled with a fluorophor. Examples offluorophores include, but are not limited to, rare earth chelates(europium chelates), Texas Red, rhodamine, fluorescein, dansyl,Lissamine, umbelliferone, phycocrytherin, phycocyanin, or commerciallyavailable fluorophors such SPECTRUM ORANGE® and SPECTRUM GREEN® and/orderivatives of any one or more of the above. Multiple probes used in theassay may be labeled with more than one distinguishable fluorescent orpigment color. These color differences provide a means to identify thehybridization positions of specific probes. Moreover, probes that arenot separated spatially can be identified by a different color light orpigment resulting from mixing two other colors (e.g., lightred+green=yellow) pigment (e.g., blue+yellow=green) or by using a filterset that passes only one color at a time.

Probes can be labeled directly or indirectly with the fluorophor,utilizing conventional methodology. Additional probes and colors may beadded to refine and extend this general procedure to include moregenetic abnormalities or serve as internal controls. By way of examplethe HER2/neu gene is in chromosome 17, and as an internal control aprobe for -satellite sequences specific for chromosome 17 (D17Z1) may beused (Vysis, Inc.) to prove diploidy in areas of non-malignant cellsand/or to establish the presence or absence of chromosome 17 aneusomy inareas of HER2/neu amplification.

After processing for FISH, the slides may be analyzed by standardtechniques of fluorescence microscopy [see for e.g. Ploem and TankeIntroduction to Fluorescence Microscopy, New York, Oxford UniversityPress (1987)]. Briefly, each slide is observed using a microscopeequipped with appropriate excitation filters, dichromic, and barrierfilters. Filters are chosen based on the excitation and emission spectraof the fluorochromes used. Photographs of the slides may be taken withthe length of time of film exposure depending on the fluorescent labelused, the signal intensity and the filter chosen. For FISH analysis thephysical loci of the cells of interest determined in the morphologicalanalysis are recalled and visually conformed as being the appropriatearea for FISH quantification.

In order to correlate cellular morphology and/or IHC with FISH, one mayuse a computer-driven, motorized stage which stores location ofco-ordinates. This may be used to evaluate the same area by twodifferent analytical techniques. For example, color images of themorphologically stained areas may be captured and saved using acomputer-assisted cooled CCD camera. The same section may besubsequently taken through the FISH procedure, the stored locationsrecalled, and the designated areas scored for the presence offluorescent nuclear signals. A similar procedure for IHC. followed byFISH is contemplated.

Typically, hundreds of cells are scanned in a tissue sample andquantification of the specific target nucleic acid sequence isdetermined in the form of fluorescent spots, which are counted relativeto the number of cells. Deviation of the number of spots in a cell froma norm (e.g., such as probing for the HER2/neu gene in a normal cellwill produce two copies, abnormal greater than two) may be indicative ofa malignancy or a predisposition to a malignancy, disease, or otherabnormality. The relative number of abnormal cells to the total cellsample population may also indicative of the extent of the condition orabnormality. In addition, using family health histories and/or geneticscreening, it is possible to estimate the probability that a particularsubject has for developing certain types of cancer. Those subjects thathave been identified as being predisposed to developing a particularform of cancer can be monitored or screened to detect early evidence ofdisease. Upon discovery of such evidence, early treatment can beundertaken to combat the disease. Similarly, those subjects who havealready developed a malignancy and who have been treated to remove thecancer or are otherwise in remission are particularly susceptible torelapse and reoccurrence, including the metastasis of tumors. Suchsubjects can be monitored and screened using the presently disclosedmethods to detect evidence of metastasis and upon discovery of suchevidence, immediate treatment can be undertaken to combat the disease.

Kits

In yet another embodiment, this invention provides a kit comprising afluorescently labeled probe specific for the target nucleic acidsequence of interest and a morphological stain and/or an antibody whichspecifically binds target antigen. The kit further comprises a set ofinstructions for applying the stain or antibody and probe to the samesection of a tissue sample. By way of example, the fluorescently labeledprobe may be complementary to the HER2/neu gene and the morphologicalstain may be HEMA 3® (CMS, Houston, Tex.). Any fluorescent label asdescribed above may be used to label the probe. The IHC/FISH kit maycomprise a fluorescently labeled probe complementary to the HER2/neugene and an antibody (e.g. polyclonal antibody) which binds to the HER2protein. The kit may include both a primary and secondary antibody,wherein the secondary antibody is conjugated to a label, e.g., anenzymatic label. The invention also provides an IHC kit which hasinstructions to perform FISH on the same section of tissue sample onwhich IHC has been previously performed.

Other optional components in the kit include one or more buffers (e.g.,block buffer, wash buffer, substrate buffer, etc), other reagents suchas substrate (e.g., chromagen) which is chemically altered by anenzymatic label, epitope retrieval solution, control samples (positiveand/or negative controls), control slide(s) etc.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tocarry out the invention and is not intended to limit the scope of whatthe inventors regard as their invention. Efforts have been made toensure accuracy with respect to numbers used (e.g., amounts,temperatures, etc.), but some experimental error and deviation should beaccounted for. Unless indicated otherwise, parts are parts by weight,molecular weight is weight average molecular weight, temperature is indegrees Centigrade, and pressure is at or near atmospheric.

Example 1 Evaluation of Morphological Stains on FISH Analysis

A sensitive and specific evaluation of breast tumors for theamplification of HER2/neu by FISH requires definitive identification andscoring of invasive ductal carcinoma cells as distinct from otherstromal and inflammatory elements found in the biopsy. Thus, it wasnecessary to identify a stain which would allow for completemorphological evaluation of the tissue, and which also allowed for easyquantification of nuclear signals upon subsequent hybridization withfluorescently labeled nucleic acid probes. Many morphological stainsfluoresce when illuminated by light of the same wavelength required tovisualize the probe fluorophores of interest. When this autofluorescenceis of a color similar to that of the probe fluorophore, signalquantification is made difficult.

Results from the evaluation of six commonly used morphological stainsmay be found in Table 2.

TABLE 2 Evaluation of Morphological Stains Autofl. Autofl. Morph. stainMorphology pre-FISH DAPI Chr 17 HER2/neu post-FISH Comments ParagonAcceptable Bright red Visible Visible Visible Bright red Redautofluoresence may interfere with FISH (Toludine blue/basic fuchsin)MGP Not Intense ND ND ND ND Red autofl. great acceptable red Gill NotMod red Visible Visible Visible Mod red Autofl. may interfere with FISHHematoxylin acceptable Mod green Weigert Acceptable Dull red VisibleVisible Visible- Mod red Post-FISH autofl. masks HER2/neu Dull green dimMethylene Acceptable Mod Visible Visible Visible Mod Possible candidateblue orange orange HEMA 3 ® Acceptable Dim green Absent Absent Absent NADestaining may have interfered with FISH (destain) HEMA 3 ® AcceptableDim green Visible Visible Visible Trace- Good candidate (w/o destain)green

Methyl green pyronin (MGP) and Gill hematoxylin were not analyzedfurther due to poor morphological definition. Paragon (toluidinebluelbasic fuchsin) and Weigert were eliminated from considerationbecause of an unacceptably high level of red or orange autofluorescencewhich masked the orange HER2/neu signals in the nucleus. Methylene blueyielded acceptable morphological staining, but demonstrated orangeautofluorescence which caused moderate difficulty in visualizingHER2/neu signals. This stain was considered a possible candidate. HEMA3® however, was superior in that it provided good morphological detailand showed only modest dim green autofluorescence which did notinterfere to any extent with FISH evaluation. An initial attempt atdestaining HEMA 3® prior to FISH was found to damage the integrity ofthe DNA in the cells and ultimately proved to be unnecessary anyway. Itwas possible for the stained tissue to be taken directly through theFISH procedure without intermediate processing, yielding high-qualitynuclear signals without any negative consequence. HEMA 3®, therefore,met all essential requirements and was the stain of choice for theproposed system. Unless otherwise indicated, the tissues and FISHanalysis were conducted as discussed in Example 2.

Example 2 Detection and Quantification of HER2/neu Amplification inBreast Tissue Materials and Methods Tissue Specimens and Preparation forFISH

Five micron sections were cut from breast tissue which had been fixed inbuffered formalin and embedded in paraffin. Sections were placed onpositively charged Superfrost Plus slides (CMS, Houston, Tex.) which hadalso been treated with poly-L-lysine or were mounted on positivelycharged slides which had no additional treatment prior to the mountingof the slides. Some material had been archived for up to fourteen years.Slide mounted tissue sections were heated on a 65° C. slide warmer forapproximately 3 seconds, placed on the bench top for 2 seconds, anddeparaffinized in Hemo-De (CMS, Houston, Tex.) for 10 minutes, ×3,followed by immersion in 100% ethanol (EtOH) for 5 minutes, ×2. Slideswere air dried in a vertical position. Those slides to be stained formorphologic evaluation were dipped in HEMA 3® Solution I (CMS, Houston,Tex.) for one second, ×3, then immediately dipped in HEMA 3® Solution IIfor one second, ×3. Each slide was rinsed in deionized, distilled waterand allowed to air dry in a vertical position. The slides were mountedand coverslipped using an aqueous-based mounting medium (BiomedaCorporation, Foster City, Calif.) for evaluation at the microscope.Areas of interest identified by the pathologist were marked on thecoverglass and subsequently circled on the underside of the slide usinga diamond-tipped pencil. The coverglass was removed by placing the slidein a water-filled Coplin jar, which had been pre-heated in a 73±2° C.waterbath, for approximately 15-30 minutes, or until the coverglassslipped off. The section was dehydrated in 70% EtOH for 3 minutes, 85%EtOH for 3 minutes, and 100% EtOH for 3 minutes. These slides, and anydeparaffinized sections that were not stained but were to be processedby FISH, were immersed in a sodium isothiocyanate Pretreatment Solution(Vysis, Inc., Downers Grove, Ill.) for 30 minutes at 80±1° C. Thesections were washed in deionized water for one minute, then in ProteaseWash Buffer (2×SSC, pH 7.4, Vysis, Inc.) for 5 minutes at roomtemperature, ×2. The sections were treated in Protease Solution (Vysis,Inc.) for 10 minutes at 37° C. Following protease digestion, thesections were washed in Protease Wash Buffer for 5 minutes, ×2, anddried on a 45-50° C. slide warmer for 2-5 minutes.

Probes

A probe spanning approximately 140 kb of the region on the long arm ofchromosome 17 containing the HER2/neu proto-oncogene (17q11.2-17q12) wasutilized to detect the presence or absence of HER2/neu amplification.Chromosome 17 aneusomy of the cell was evaluated using an -satellitesequence probe (D17Z1) specific for the centromere of chromosome 17 (CEP17). The probes were provided by Vysis, Inc. in an optimized cocktailmixture where the HER2/neu probe is directly labeled with thefluorophore SPECTRUM ORANGE® (peak excitation/emission=559/588) and theCEP 17 probe is directly labeled with the fluorophore SPECTRUM GREEN®(peak excitation/emission=509/538)(Vysis, Inc.).

In situ Hybridization

The pre-treated (see above) slide-mounted sections were immersed inDenaturation Solution (70% formamide/2×SSC, pH 7.0) for 5 minutes at73±1° C. Sections were then dehydrated in 70% EtOH for one minute, 85%EtOH for one minute, and 100% EtOH for one minute. Drained slides wereplaced on a 45-50° C. slide warmer for 2-5 minutes just beforeapplication of the hybridization mixture. This mixture was prepared asdirected by VYSIS® protocol. The probe solution was denatured byincubation in a 73° C. waterbath for 5 minutes. Following briefcentrifugation, 10 L of the solution was pipetted onto each section, acoverslip applied, and the edges sealed with rubber cement.Hybridization was carried out in a humidified box overnight (14-18 h) ina 37° C. incubator. The rubber cement was carefully removed and theslides inmmersed in room temperature Wash Buffer I (0.4×SSC/0.3% NP-40)until the coverslip floated off. Slides were drained of excess liquid byblotting the edges on a paper towel, then placed in a Coplin jar of WashBuffer I, which had been prewarmed to 72±1° C., for 2 minutes. Sectionswere washed in room temperature Wash Buffer II (2×SSC/0.1% NP-40) forone minute, drained vertically on paper towels, and allowed to air-dryin darkness being careful not to overdry. Once slides were dry, 15 L ofDAPI counterstain (a mixture of equal parts DAPI I and DAPI II, Vysis,Inc.) was pipetted onto the hybridization area. A coverglass was addedand the edges sealed with clear nail polish. Slides were analyzedimmediately or stored in the dark at −20° C.

Controls

Cultured cell lines SKBR3, MDA175, and MDA231 were harvested,formalin-fixed, and paraffin-embedded for use as highly amplified,barely amplified, and non-amplified controls, respectively. These celllines were used in the evaluation of the morphological stains. Thepresence of appropriate nuclear FISH signals served to, assure that thereagents and procedure involved in the devised methodology workedproperly.

Microscopy

An Olympus BX60 epifluorescence microscope equipped with a 100 wattmercury-arc lamp, a 40×UPlanApo objective, and a 100×UPlanFl objectivewas used. The filter slider was built by Chroma Technology Corporation(Brattleboro, Vt.) for Olympus and included three single band passfilters optimized for visualization of DAPI, FITC, and Texas Redfluorochromes, a dual-band pass filter optimized for FITC and Texas Red,and a triple-band pass filter optimized for DAPI, FITC, and Texas Red.The microscope was also equipped with 4×, 10×, 20×, and 40× UPlanFlobjectives for use in transmittance light microscopy. A BioPoint X,Y(Ludl Electronic Products, Ltd., Hawthorne, N.Y.) computer-drivenmotorized stage was used for storage and retrieval of physicallocations. Images were acquired using a CoolCam 2000 3-chip color cooledCCD camera (Sci-Measure, Atlanta, Ga.) and printed using a TektronixPhaser 440 dye sublimation printer (Tektronix, Inc., Wilsonville,Oreg.).

FISH Scoring Criteria

After FISH processing, tissue sections were scanned using the 40×objective to evaluate for tissue loss and to ensure that hybridizationwas uniform across the section. Morphologically identified areas ofnormal or tumor tissue were located on FISH processed slides byrecalling stored x and y coordinates. Alternatively, if locationcoordinates had not been stored, a saved morphological image (HEMA 3®stain) of the area of interest was used as a reference to localize theappropriate area after FISH processing. Signals were enumerated usingthe 100× objective, switching between different filters to optimizesignal discrimination. Nuclear boundaries were defined by DAPIexcitation. Only those nuclei were scored which could be clearlyidentified as intact and non-overlapping. The presence of nuclei havingno CEP 17 or no HER2/neu signaled the possibility of a hybridizationfailure or the existence of truncated nuclei, suggesting an unacceptablythin section. The hybridization quality of the entire section wasassessed completely before making a decision regarding acceptability. Incases of high HER2/neu gene amplification (>10-20 signals per nucleus),signals were often coalesced into clusters and could not be preciselyenumerated. If, as in some instances, the CEP 17 signals appearedfragmented, a broken signal was scored as two separate signals if thedistance between them was of sufficient size that a third comparablesignal could be passed through it. A minimum scoring goal of 100 nucleiper area was attempted; however, in certain circumstances, it was notpossible to meet this goal. For example, areas of normal tissue tendedto be minimally represented and relatively non-cellular; in thesesituations, all clearly discernible cells were scored. In areas ofhighly amplified HER2/neu, a total of 30-40 cells was consideredadequate to establish amplification status.

Immunohistochemical Staining

Sections cut serially from those used for FISH analysis wereimmunohistochemically stained using a murine monoclonal antibody againstHER2 [4D5; Fendly et al. Cancer Research 50:1550-1558 (1990)].Slide-mounted sections were placed into xylenes 3×, 10 minutes eachtime, then dehydrated in absolute ethanol 2×, 5 minutes each. Endogenousperoxidase activity was quenched by placing the slides in a 0.3%solution of hydrogen peroxide in methanol. The sections were thenserially rehydrated in 95% ethanol for 5 minutes, followed by 80%ethanol for 5 minutes. Sections were incubated briefly in the bufferroutinely used with the Ventana ES automated imniunostaining instrument.The staining program began with a 4 minute protease treatment.Incubation with the primary antibody (10 μg/ml) proceeded for themaximum allowable time period, 32 minutes. The detection system employeda biotin/avidin reaction using DAB and a hematoxylin counterstain. Afterstaining and detection, the sections were dehydrated in a graded seriesof ethanol into xylenes in preparation for permanent coverslipping.

Immunohistochemistry Scoring Criteria

The immunohistochemical staining was interpreted by a breast tumorpathologist and the results assigned to one of four categories definedin the following way: negative/weak, cytoplasmic, 1+, 2+. The secondcategory was reserved for those cells in which staining occurred only inthe cytoplasm. The latter two categories applied to cell surfaceantibody staining. Images of immunostained sections representative ofeach category were captured using the CCD camera. Attempts were made torelocate areas that had been scored for FISH on the corresponding serialsection, and whenever possible that image was captured.

Specimens

Formalin-fixed, paraffin-embedded human breast tissue was obtainedthrough clinical submission of specimens to Cytometry Associates andVanderbilt University Medical Center (VUMC), Department of Pathology.Some materials may have been archived at VUMC for up to fourteen years.In some cases, the specimens were identified for the study based on thelikelihood of HER2 amplification as predicted by IHC results or hormonereceptor status. Other cases were known to be disease-free based onmorphological evaluation at VUMC.

Stain Plus FISH vs. FISH Only

Initially, the effect of HEMA 3® stain on the ability to score FISH wasevaluated in formalin-fixed, paraffin-embedded cells harvested from eachof three cell-lines of known HER2/neu amplification status (FIG. 1).Results from analysis by two individuals were comparable andsuccessfully identified the different cell-lines as being of theappropriate, expected amplification status. The stain had no adverseeffect on FISH quantification in these cell lines. A total of 10 patientspecimens was included in the study comparing stained and hybridizedtissue versus tissue which was hybridized without prior staining. Thesame areas of tumor and normal cells identified on each of two serialsections was analyzed for the presence of HER2/neu and chromosome 17 byFISH either with or without prior morphological staining. Two analystsperformed FISH quantification of the designated areas. MeanHER2/neu:Chr17 ratios for each of the areas of tumor in the ten patientspecimens scored, plus the three cell lines, are shown in FIG. 2. Therewas no statistically significant quantitative difference as a result ofthe two different treatments of the tissue sections in either the tumorareas (P=0.196) or the normal areas (P=0.597) scored. The differences inthe mean values between the two analysts performing FISH quantificationwere not statistically significant in areas of either normal cells(P=0.065) or tumor cells (P=0.459). In addition, the inter-observereffect did not depend on which treatment the tissue section hadundergone, whether in areas of normal morphology (P=0.513) or in areasof invasive carcinoma (P=0.971).

Blinded vs Non-Blinded Study

A study was designed to determine whether prior knowledge ofmorphological cell type biased the scoring of FISH signals. Ten patientspecimens included in the study were stained with HEMA 3® formorphological evaluation by a pathologist. Areas of normal cellulartissue and areas of invasive carcinoma were identified, their x/ycoordinates stored, and images captured and saved. The files were namedand stored in an anonymous and random manner so that the two analystswho scored the areas for FISH signals were unaware of their identity orcell type. After the blinded evaluation was completed, the analysts weregiven a second list of file names of the same ten images and locationsof normal and tumor areas, but in this case, the identity and cell typewas provided. To score FISH signals; the analyst used the x/y coordinateprovided to relocate the area of interest and used the saved image toconfirm accurate relocation. The mean HER2/neu:Chr 17 ratio wasdetermined for each area scored by the analysts, and the mean ratio ofthe two analysts calculated. Mean ratios for blinded vs non-blindedassessment of both normal tissue and tumor tissue are shown in FIGS. 3and 4, respectively.

Non-diseased Normal vs Patient Normal

Normal range data were generated from analysis of histologically normalbreast tissue obtained from mammoplasty surgical procedures. Tenbiopsies were evaluated by two analysts for quantitative detection ofHER2/neu and chromosome 17 by FISH. The mean HER2/neu:Chr 17 ratio forthe two analysts was 1.07. In order to establish a normal range, areasof normal cellular tissue were identified in 38 biopsies of breasttissue from patients diagnosed with invasive carcinoma. The mean ratiofor these specimens was also 1.07. Statistical treatment of the datawith a two way repeated measures ANOVA found no significant differencebetween the two tissue types (P=0.821). Normal range values (≧0.9 and≦1.2) were established based on the diseased normal data, setting upperand lower limits by calculating two standard deviations from the mean(Table 3).

TABLE 3 Normal Range Determinations Mean Range Tissue Ratio n 1 SD 2 SD3 SD Non-disease 1.07 20 1.06 1.09 1.04 1.11 1.02 1.13 Disease 1.07 780.99 1.15 0.91 1.23 0.83 1.32

Definition of a normal range creates a threshold for determiningamplification status for HER2/neu. In addition, correlation betweennormal values derived from non-diseased and diseased tissue providesjustification for use of a morphologically normal cellular area in atumor biopsy as an internal biological control for specificity in thatspecimen.

Patient Cohort

Using the described system combining morphological staining and FISH, atotal of 46 cases of known invasive breast carcinoma were evaluated.Serial sections from each were stained immunohistochemically with the4D5 antibody to HER2/neu for comparative purposes. HER2/neu andchromosome 17 fluorescent nuclear signals were scored by two analystsand the mean ratios calculated. Ratios for HER2/neu amplification wereassigned to one of two ranges, either moderately amplified borderline orhighly amplified. The lower limit of the moderate amplification range(1.3) was established statistically by determining at what value theratio differed significantly from normal. The upper limit for moderatelyamplified status (2.0) was taken from previously published data whichdefined true amplification as having a ratio of >2.0 [Pauletti et al.Oncogene 13:63-72 (1996)]. The relationship between the scoring systemsused for HER2/neu amplification by FISH and overexpression by IHC isdepicted in Table 4.

TABLE 4 Scoring System for HER2 Amplification/Expression HER2Amplification/Expression IHC score FISH ratio Negative Negative 0.9-1.2Borderline 1+ 1.3-2.0 Amplified 2+ >2.0

Immunohistochemistry scores were based on membrane staining only;specimens which exhibited cytoplasmic staining (n=4) were excluded fromthe study data. Correlations between FISH mean ratios andimmunohistochemistry scores showed a positive correlation coefficient(0.760) and a low P value (<0.001). Both techniques identifiedunambiguous amplification or overexpression of HER2/neu equally wellwith two exceptions (FIG. 5). One case which produced an IHC score of 2+fell into the moderately amplified range by FISH (ratio=1.7), and onecase which showed high amplification by FISH (ratio=9.8) had an IHCscore of 1+. FISH quantification of HER2/neu placed ten specimens in themoderate amplification range. Immunohistochemical evaluation identifiedonly four specimens showing moderate positivity (score of 1+),suggesting a less than optimal sensitivity associated with the hum4D5-8antibody.

Although the present invention has been described in some detail by wayof illustration for the purposes of clarity of understanding, it will beapparent that certain changes and modifications may be practiced withinthe scope of the appended claims. Such modifications which may beapparent to a person skilled in the art are intended to be within thescope of the invention.

1. A method of correlating cellular morphology with the presence of acellular target nucleic acid sequence in a section of a tissue samplecomprising the following steps: (a) staining the section of tissuesample with a morphological stain; (b) determining cellular morphologyin the section of tissue sample; (c) hybridizing a first labeled nucleicacid probe to the target nucleic acid sequence in the same section oftissue sample, wherein said label is selected from the group of labelsconsisting of radioisotopes, colloidal gold particles, fluorescentlabels, and enzyme-substrate labels; (d) detecting the presence of thefirst nucleic acid probe in the section of tissue sample; and (e)correlating step (b) with step (d).
 2. The method of claim 1, whereinthe first nucleic acid probe is constructed to hybridize to the targetnucleic acid sequence indicating a genetic abnormality selected from thegroup consisting of amplification, addition, substitution, translocationand deletion.
 3. The method of claim 1, wherein amplification of thetarget nucleic acid sequence is determined in step (d).
 4. The method ofclaim 3, wherein the target nucleic acid sequence is HER2/neu gene. 5.The method of claim 1, wherein the morphological stain used in step (a)does not significantly autofluoresce at the same wavelength as afluorescent label of the first nucleic acid probe.
 6. The method ofclaim 1, further comprising hybridizing a second labeled nucleic acidprobe to a nucleic acid sequence in the section of tissue sample,wherein the second nucleic acid probe comprises a label distinguishablefrom a label of the first nucleic acid probe.
 7. The method of claim 6,wherein the second nucleic acid probe determines chromosome copy number.8. The method of claim 1, wherein the morphological stain used in step(a) comprises xanthine dye and thiazine dye.
 9. The method of claim 1,wherein the morphological stain comprises methylene blue, a xanthinedye, or a thiazine dye.
 10. The method of claim 1, wherein the tissuesample is selected from the group consisting of breast, prostate, ovary,colon, lung, endometrium, stomach, salivary gland and pancreas tissuesample.
 11. The method of claim 1, wherein the target nucleic acidsequence is selected from the group consisting of HER2/neu gene and thecentromere of chromosome
 17. 12. The method of claim 1, wherein thefirst nucleic acid probe is labeled with a fluorescent label selectedfrom the group consisting of derivatives of Texas Red, fluorescein,phycocrytherin, rhodamine, phycocyanin, dansyl, umbelliferone, a greenfluorophore, and an orange fluorophore.
 13. The method of claim 1,wherein the section of tissue sample is not destained prior to step (c).14. The method of claim 1, wherein the section of tissue sample isobtained from fixed, paraffin-embedded tissue sample.
 15. A method ofcorrelating cellular morphology and the presence of a cellular targetprotein with the presence of a cellular target nucleic acid sequence ina section of a tissue sample comprising the following steps: (a)staining the section of tissue sample with a morphological stain; (b)determining cellular morphology in the section of tissue sample; (c)contacting the section of sample tissue with an antibody whichspecifically binds to the target protein; (d) determining binding of theantibody to the section of tissue sample; (e) hybridizing a labelednucleic acid probe to the target nucleic acid sequence in the samesection of tissue sample, wherein said label is selected from the groupof labels consisting of radioisotopes, colloidal gold particles,fluorescent labels, and enzyme-substrate labels; (f) detecting thepresence of the labeled nucleic acid probe in the section of tissuesample; and (g) correlating step (b) and (d) with step (f), wherein themorphological stain used in step (a) does not significantly interferewith detection of the labeled nucleic acid probe used in step (e).
 16. Akit comprising: (a) a morphological stain; (b) a labeled probecomplementary to a genetic abnormality in a cellular target nucleic acidsequence, where a genetic abnormality comprises a deletion,substitution, addition, translocation, or amplification of a nucleicacid sequence relative to the normal native cellular target nucleic acidsequence, and wherein said label is selected from the group of labelsconsisting of radioisotopes, colloidal gold particles, fluorescentlabels, and enzyme-substrate labels; (c) a primary antibody whichspecifically binds a cellular target protein; (d) a labeled secondaryantibody which specifically binds to the primary antibody; and (e)instructions for applying the stain (a) and probes (b), (c) and (d) tothe same section of tissue sample wherein autofluorescence of themorphological stain does not significantly interfere with detection ofthe labeled nucleic acid probe.
 17. The kit of claim 16, wherein thenucleic acid probe hybridizes to HER2/neu nucleic acid.
 18. The kit ofclaim 16, wherein the morphological stain comprises methylene blue, axanthine dye, or a thiazine dye.
 19. The kit of claim 16, wherein thesecondary antibody is labeled with an enzymatic label which catalyzeschemical alteration of a substrate compound.
 20. The kit of claim 16,wherein the nucleic acid probe hybridizes to HER2/neu nucleic acid andthe primary antibody specifically binds to HER2 protein.
 21. The kit ofclaim 20, further comprising instructions describing methods forapplying the probe to a section of tissue sample having a score of 1+ or2+ for HER2 Protein Staining Intensity and for analyzing the results ofsaid application.
 22. The method of claim 1, wherein the first nucleicacid probe is labeled with a radioisotope label selected from the groupconsisting of ³⁵S, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I.
 23. The method of claim 1,wherein the first nucleic acid probe is labeled with colloidal goldparticles.
 24. The method of claim 1, wherein the first nucleic acidprobe is labeled with an enzyme substrate label selected from the groupof enzyme substrate labels consisting of horseradish peroxidase (HRPO),alkaline phosphatase (AP), and β-D-galactosidase (β-D-Gal).
 25. Themethod of claim 15, wherein the first nucleic acid probe is labeled witha radioisotope label selected from the group consisting of ³⁵S, ¹⁴C,¹²⁵I, ³H, and ¹³¹I.
 26. The method of claim 15, wherein the firstnucleic acid probe is labeled with colloidal gold particles.
 27. Themethod of claim 15, wherein the first nucleic acid probe is labeled withan enzyme substrate label selected from the group of enzyme substratelabels consisting of HRPO, AP, and β-D-Gal.
 28. The method of claim 15,wherein the first nucleic acid probe is labeled with a fluorescent labelselected from the group consisting of derivatives of Texas Red,fluorescein, phycocrytherin, rhodamine, phycocyanin, dansyl,umbelliferone, a green fluorophore, and an orange fluorophore.
 29. Thekit of claim 16, wherein the first nucleic acid probe is labeled with aradioisotope label selected from the group consisting of ³⁵S, ¹⁴C, ¹²⁵I,³H, and ¹³¹I.
 30. The kit of claim 16, wherein the first nucleic acidprobe is labeled with colloidal gold particles.
 31. The kit of claim 16,wherein the first nucleic acid probe is labeled with an enzyme substratelabel selected from the group of enzyme substrate labels consisting ofHRPO, AP, and β-D-Gal.
 32. The kit of claim 16, wherein the firstnucleic acid probe is labeled with a fluorescent label selected from thegroup consisting of derivatives of Texas Red, fluorescein,phycocrytherin, rhodamine, phycocyanin, dansyl, umbelliferone, a greenfluorophore, and an orange fluorophore.
 33. The method of claim 15,wherein the first nucleic acid probe is labeled with a fluorescentlabel, further comprising the step of using a filter set that passesonly one color at a time.
 34. The kit of claim 16, wherein the firstnucleic acid probe is labeled with a fluorescent label, the kit furthercomprising a filter set having a filter that passes only one color at atime.
 35. A method for detecting the presence of a cellular targetprotein and a cellular target nucleic acid in a tissue sample,comprising the steps of: (a) contacting a section of the tissue samplewith an antibody which specifically binds to the target protein; (b)determining binding of the antibody to the section of tissue sample; (c)hybridizing a labeled nucleic acid probe to the target nucleic acid inthe same section of tissue sample; (d) detecting the presence of thelabeled nucleic acid probe in the section of tissue sample; (e)comparing the results of step (b) with the results of step (d); and (f)staining the section of tissue sample with a morphological stain. 36.The method of claim 35 wherein the label is a fluorescent dye.
 37. Themethod of claim 35, wherein the nucleic acid probe is constructed tohybridize to the target nucleic cid sequence indicating a geneticabnormality selected from the group consisting of amplification,addition, substitution, translocation and deletion.
 38. The method ofclaim 35, wherein the target nucleic acid is a HER2/neu gene.
 39. Themethod of claim 35, wherein the target protein is a HER2 protein. 40.The method of claim 35, wherein the tissue sample is selected from thegroup consisting of breast, prostate, ovary, colon, lung, endometrium,stomach, salivary gland and pancreas tissue samples.
 41. The method ofclaim 35, wherein the section of tissue sample is obtained from fixed,paraffin-embedded tissue sample.
 42. The method of claim 35 wherein thestaining is performed before step (a).
 43. The method of claim 35wherein the staining is performed with a morphological stain comprisingmethylene blue, xanthine dye, or thiazine dye.
 44. The method of claim43 wherein the morphological stain comprises xanthine dye and thiazinedye.